Protein factors and hormones involved in human health care have been currently produced by pharmaceutical industry by extraction or by recombinant technology in the last decades. Expression of genetic constructs involving the desired genes were successfully expressed in bacteria, yeast or mammalian cell lines. However, the use of mammalian cell cultures to obtain complex proteins, such as those which require a proper glycosylation pattern, involves high cost procedures.
Recombinant DNA technology has been used increasingly over the past decade for the production of commercially important biological materials. To this end, the DNA sequences encoding a variety of medically important human proteins have been cloned. These include insulin, plasminogen activator, alpha1-antitrypsin and coagulation factors VIII and IX. At present, even with the emergent recombinant DNA techniques, these proteins are usually purified from blood and tissue, an expensive and time consuming process which may carry the risk of transmitting infectious agents such as those causing AIDS and hepatitis.
Although the expression of DNA sequences in bacteria to produce the desired medically important protein looks an attractive proposition, in practice the bacteria often prove unsatisfactory as hosts because in the bacterial cell foreign proteins are unstable and are not processed correctly.
Recognizing this problem, the expression of cloned genes in mammalian tissue culture has been attempted and has in some instances proved a viable strategy. However, batch fermentation of animal cells is an expensive and technically demanding process.
There is therefore a need for a high yield, low cost process for the production of biological substances such as correctly modified eukaryotic polypeptides. The absence of agents that are infectious to humans would be an advantage in such a process.
The possibility of obtaining transgenic animals, like cattle, for a desired gene, with the aim of getting large amounts of a human protein in milk, has been of great interest to the industry. Several groups in the literature report their success on producing human serum albumin, alpha anti-trypsin, and some other examples in transgenic cows or goats.
Many experiments have been previously performed in mice or rats, and transgene expression was always preferred to be confined to the mammary glands since beta casein or lactalbumin promoters were employed, which respond only to mammary gland transcription factors in lactating females.
The expression of a heterologous protein exclusively in milk is meant to avoid undesired influence on the host animal health and provide an easy method for purification.
People are now devoted to set up several systems to improve the yield of cell transfection or selection, and choose the source of homologous fetal somatic cell to improve survival and immunity conditions of cloned animals.
On the other hand, there is enormous interest in somatic cell nuclear transfer, mainly to make possible the propagation of elite domestic animals and engineering of transgenic animals, for agricultural and biomedical purposes. Briefly, nuclear transfer (NT) involves the enucleation of a recipient oocyte, followed by the transfer of donor cell to the perivitelline space in close apposition of the recipient cytoplast, and their fusion. Development is induced artificially by chemical or physical activation. Production of cloned offspring by somatic cell nuclear transfer has been successfully attained in sheep (Campbell, K. H., et al., Nature 380: 64-66 (1996), 1996; Wells, D. N., et al., Biol Reprod 57: 385-393 (1997); Wilmut, I., et al, Nature 385: 810-813 (1997)); goat (Baguisi, A., et al., Nat Biotechnol 17: 456-461 (1999)) and in cow (Cibelli, J. B., et al., Science 280: 1256-1258 (1998); Kato, Y., et al., Science 282: 2095-2098 (1998); Wells, D. N., et al., Reprod Fertil Dev 10: 369-378 (1998)).
There are several factors that influence the results of NT including the methods of enucleation, fusion, activation and donor-recipient cell cycle synchrony. High efficiencies in enucleation of recipient oocytes have been achieved using DNA specific vital dyes to visualize chromatin (Stice, S. L., and Keefer, C. L., Biol Reprod 48: 715-719 (1993); Westhusin, M. E., et al., J Reprod Fertil 95: 475-480 (1992)). Fusion of the donor cell with the recipient oocyte depends on the accuracy of cell alignment in the pulse field, contact of the donor cell with the recipient oocyte and size of the donor cells (Collas, P., et al., Anal Biochem 208: 1-9 (1993)). Activation of NT reconstructed embryo has been refined and rates of development to blastocysts are equivalent to in vitro fertilized oocytes (Liu, L., et al., Mol Reprod Dev 49: 298-307 (1998)).
Successful development of NT embryos has been accomplished using mature oocytes (Willadsen, S. M., Nature 320: 63-65 (1986)), zygotes (McGrath, J., and Solter, D., Dev Biol N Y 4: 37-55 (1985)), and cleavage-stage embryos (Tsunoda, Y., et al., J Reprod Fertil 96: 275-281 (1992)) as recipient cytoplasts; however, this is dependent on the source of the donor nucleus. Compatibility of the cell cycle between the recipient cytoplasts and the donor cells is one of the important factors that influence the development of NT embryos. Appropriate synchronization is necessary to preserve the ploidy of the reconstituted embryo.
The mitotic cell cycle has the following consecutive phases: pre-replication gap (G1), synthesis of DNA (S), pre-mitotic gap (G2) and mitosis (M). During a single cell cycle, all genomic DNA replicates once prior to mitosis. An interphase donor nucleus transferred into an enucleate mature oocyte (metaphase II) undergoes several morphological changes. After fusion, but prior to donor nuclear envelope breakdown (NEBD), the chromosome condenses (PCC). These changes are induced by the activity of maturation/mitosis/meiosis-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) (Collas, P., and Robl, J. M., Biol Reprod 45: 455-465 (1991)). MPF and MAPK activities are found in all meiotic and mitotic cells and are highest at metaphase and in mammalian oocytes these high levels also induce arrest in metaphase II. Reduction of MPF and MAPK by fertilization or activation with calcium ionophore is the signal for completion of meiosis, second polar body emission, sperm nucleus decondensation and pronuclear formation.
The direct effect of NEBD and PCC on donor chromatin is dependent on the cell cycle of the donor nucleus at the time of the transfer. Diploid G0/G1 nuclei condense to form single chromatids, but tetraploid G2 nuclei condense to form double chromatids. However, nuclei in S phase at the time of the transfer show a characteristic “pulverized” appearance; PCC produces extensive DNA damage. Therefore, correct ploidy can be produced by transferring a G1 or G0 nuclei into metaphase II oocytes at the time of activation or before. A second method is to transfer nuclei in previously activated oocyte, in S phase, in this case is possible to use a donor cell in G1, G0 or S phase. Because MPF and MAPK are low; the chromatin decondenses, and undergoes DNA replication without PCC and NEBD.
A third synchronization scheme has been reported in mice, where development of a live offspring was produced by embryo reconstruction using a G2 or metaphase donor cell and an enucleated metaphase 2 oocyte (Cheong, H. T., et al., Biol Reprod 48: 958-963 (1993); Kwon, O. Y., and Kono, T., Proc Natl Acad Sci USA 93: 13010-13013 (1996)). The extrusion of a polar body from the NT reconstructed embryo was reported, resulting in single diploid embryo and a diploid polar body (Kwon, O. Y., and Kono, T., Proc Natl Acad Sci USA 93: 13010-13013 (1996)). However, there is no report of polar body formation after NT into enucleated MII oocytes in cattle, sheep or pigs, suggesting differences between species in the mechanics controlling formation of intact spindles and extrusion of polar bodies.
The cell cycle stages of the donor cell and the recipient have been suggested to be also important to reprogram the donor cell nuclei. Increasing the time between donor nuclei transfer and zygotic transcription may improve nucleus reprogramming. For this reason, several authors activated the oocyte several hours after fusion (Cibelli, J. B., et al., Science 280: 1256-1258 (1998); Wakayama, T., et al., Nature 394: 369-374 (1998); Wells, D. N., et al., Biol Reprod 60: 996-1005 (1999)). Other reports applied sequential nuclear transfer (Stice, S. L., and Keefer, C. L., Biol Reprod 48: 715-719 (1993)).
An unexplored procedure to increase the time of donor nucleus reprogramming is by nuclear transfer before metaphase II. After germinal vesicle breakdown (GVBD), all the nuclei events are regulated by a substantial increase in oocyte cytosolic MPF and MAPK, which prevent reconstruction of the nuclear envelope and entrance in the S phase until fertilization or activation. Therefore, a maturing oocyte may be a universal recipient for metaphase or G2 donor cell. Even G1 or G0 can be used as donor cells if activation induces an S phase before cell division.
When blastomeres in G2 or M are used as donor cells, nuclear reprogramming is possible (Cheong, H. T., et al., Biol Reprod 48: 958-963 (1993); Kwon, O. Y., and Kono, T., Proc Natl Acad Sci USA 93: 13010-13013 (1996); Liu, L., et al., Mol Reprod Dev 47: 255-264 (1997)). One explanation is that some factors are displaced from the chromatin as a result of chromosome condensation. In fact, for nuclear transfer, NEBD and PCC have been considered morphological signs of nucleus reprogramming. Additionally, at the time of fertilization sperm chromatin is extremely condensed, and its volume is considerably smaller than that of nuclei of somatic cells, and oocyte has the ability to remove sperm nuclear protein. Oocyte chromosomes, during sperm-oocyte fusion, are also condensed. It is possible that condensed chromatin conformation may have some biological relevance. Consequently, by mimicking this situation by metaphase nuclear transfer, a metaphase-enucleated recipient could improve NT result. However, few researchers have used this approach in domestic animals and using blastomeres as donor cells (Liu, L., et al., Mol Reprod Dev 47: 255-264 (1997)).
One goal of this invention is to characterize and refine existing somatic cell nuclear transfer to a reliable and economical technique to produce genetically identical calves from adult donor cells.